The present invention pertains to reagents and methods for producing virus vectors, in particular, reagents and methods for producing adeno-associated virus vectors.
Adeno-associated virus (AAV) type 2 is a nonpathogenic human parvovirus that generally depends on coinfection with a helper virus (adenovirus or herpesvirus) for efficient replication (reviewed in Berns (1996). Parvoviridae: The viruses and their replication, p. 2173-2197, in B. N. Fields (ed.), Fields Virology, 3rd ed., vol. 2, Raven, Philadelphia). The linear, single-stranded DNA genome of AAV encodes two open reading frames (rep and cap) flanked by 145 bp inverted terminal repeats (ITR) (Srivastava et al., (1983) J. Virol. 45:555). Replication of the AAV genome requires two viral components, the ITR that serves as the origin of replication (Hauswirth et al., (1977) Virology 78:488; Straus et al., (1976) Proc. Natl. Acad. Sci. USA 73:742; Samulski et al., (1983) Cell 33:135; Senepathy et al., (1984) J. Mol. Biol. 179:1) and the rep gene products (Senepathy et al., (1984) J. Mol. Biol. 179:1, Hermonat et al., (1984) J. Virology 51:329; Tratschin et al., (1984) J. Virology 51:611). The rep gene encodes four multifunctional proteins (Hermonat et al., (1984) J. Virology 51:329; Tratschin et al., (1984) J. Virology 51:611; Mendelson et al., (1986) J. Virology 60:823; Trempe et al., (1987) Virology 161:18) that are expressed from two promoters at map units 5 (p5) and 19 (p19). The larger Rep proteins transcribed from the p5 promoter (Rep78 and Rep68), are essentially identical except for unique carboxy termini generated from unspliced (Rep78) and spliced (Rep68) transcripts, respectively (Srivastava et al, (9183) J. Virol. 45:555). Two smaller rep proteins (Rep52, Rep40), transcribed from the p19 promoter are amino terminal truncations of Rep78 and Rep68, respectively.
Several biochemical activities of Rep78 and Rep68 have been characterized as necessary for AAV replication. These include specific binding to the AAV ITR (Ashktorab et al., (1989) J. Virology 63:3034; Im et al., 1989) J. Virology 63:3095; Snyder et al., (1993) J. Virology 67:6096) and site-specific endonuclease cleavage at the terminal resolution site (trs) (Im et al., (1990) J. Virology 63:447; Im et al., (1992) J. Virology 66:1119; Snyder et al., (1990) Cell 60:105; Snyder et al., (1990) J. Virology 64:6204). Rep78/68 also possess ATP dependent DNA-DNA helicase ((Im et al., (1990) J. Virology 63:447; Im et al., (1992) J. Virology 66:1119) and DNA-RNA helicase as well as ATPase activities (Wonderling et al., (1995) J. Virology 69:3542). In addition to these activities required for replication, Rep78/68 also regulate transcription from the viral promoters (Beaton et al., (1989) J. Virology 63:4450; Labow et al., (1986) J. Virology 60:251; Tratschin et al., (1986) Mol. Cellular Biol. 6:2884; Kyostio et al., (1994) J. Virology 68:2947; Pereira et al., (1997) J. Virology 71:1079), and have been shown to mediate viral targeted integration (Xiao, W., (1996), xe2x80x9cCharacterization of cis and trans elements essential for the targeted integration of recombinant adeno-associated virus plasmid vectorsxe2x80x9d, Ph.D. Dissertation, University of North Carolina-Chapel Hill; Balague et al., (1997) J. Virology 71:3299; LaMartina et al., (1998) J. Virology 72:7653; Pieroni et al., (1998) Virology 249:249).
Mutant studies of the Rep proteins have indicated that the activities of Rep can be divided into partially distinct functional domains (FIG. 1A) that are spread throughout the protein (Chejanovsky et al., (1989) Virology 173:120; McCarty et al., (1992) J. Virology 66:4050; Yang et al., (1992) J. Virology 66;6058; Owens et al., (1993) J. Virology 67:997; Weitzman et al., (1996) J. Virology 70:2440; Walker et al., (1997) J. Virology 71:2722; Walker et al., (1997) J. Virology 71:6996; Davis et al., (1999) J. Virology 73:2084; Urabe et al., (1999) J. Virology 73:2682). These include regions required for binding to the ITR; a putative NTP-binding/ATPase domain, nuclear localization and residues putatively required for nicking and helicase functions. Several mutations within the NTP-binding/ATPase domain that lacked trs endonuclease and viral replication were also defective for trans-activation functions suggesting a need for further mutant analysis (McCarty et al., (1992) J. Virology 66:4050). Since most mutants disrupt multiple Rep mediated functions for the AAV life cycle, detailed characterization of distinct functions has been difficult (McCarty et al., (1992) J. Virology 66:4050; Yang et al., (1992) J. Virology 66:6058; Owens et al., (1993) J. Virology 67:997; Weitzman et al., (1996) J. Virology 70:2440; Walker et al., (1997) J. Virology 71:2722; Walker et al., (1997) J. Virology 71:6996; Davis et al., (1999) J. Virology 73:2084; Urabe et al., (1999) J. Virology 73:2682).
One of the considerations in designing methods for production or delivery of AAV is the toxicity of the AAV Rep proteins to helper viruses (e.g., adenovirus). Thus the AAV rep/cap genes are typically provided on a separate vector from the helper virus or silenced in the cell chromosome. In addition, the rep gene products are frequently cytotoxic to the cells used to package rAAV vectors.
The use of temperature sensitive (ts) mutations has proven to be an effective method for elucidating the essential functions of viral proteins (Murphy et al., (1988) Virus Research 11:1; Crowe et al., (1996) Virus Genes 13:269; Rhode (1978) J. Virology 25:215; Burns et al., (1992) Virology 189:568). One approach for generating ts mutants has been to utilize the charged-to-alanine mutagenesis strategy (Cunningham et al., (1989) Science 244:1081; Bennett et al., (1991) J. Biological Chemistry 266:5191; Bass et al., (1991) Proc. Nat. Acad. Sci. USA 88:4498; Wertman et al., (1992) Genetics 132:337; Diamond et al., (1994) J. Virology 68:863; Parkin et al., (1996) Virus Research 46:31). The rationale of this approach is that since most charged residues are found on the protein surface they are expected to exert little effect on protein folding or stability (Cunningham et al., (1989) Science 244:1081; Wertman et al., (1992) Genetics 132:337; Dao-Pin et al., (1991) Biochemistry 30:11521), but could feasibly make a protein more thermosensitive by disrupting electrostatic and H-bonding interactions (Diamond et al., (1994) J. Virology 68:863). This technique does not always yield ts proteins, but is a popular approach when the crystal structure of the protein in question is lacking.
Accordingly, ts AAV Rep mutants would be advantageous to provide a functional Rep protein that may be controlled or inactivated at non-permissive temperatures so that the toxicity normally associated with AAV Rep proteins may be diminished or avoided.
The present invention addresses a need in the art for improved strategies for producing AAV vectors. In addition, the present invention is directed to a need in the art for improved reagents and methods for gene delivery.
Current techniques for packaging AAV vectors are not readily amenable to large-scale production. These difficulties arise, in part, from toxicity of the AAV Rep proteins to helper viruses and host cells, thus requiring that the AAV rep/cap genes be provided on a separate vector from the helper virus or silenced in the cell chromosome. In addition, the rep gene products are frequently cytotoxic to the cells used to package rAAV vectors. The present invention provides temperature-sensitive (ts) AAV Rep proteins, and nucleotide sequences encoding the same, that may be used according to the methods disclosed herein to reduce or mitigate the problems posed by Rep protein toxicity. The activity of the inventive ts Rep proteins may be readily controlled by shifting the cells packaging the rAAV vectors to permissive or non-permissive temperatures as desired.
As one aspect, the present invention provides a ts AAV Rep protein. The ts Rep protein may be any of the AAV Rep proteins, but is preferably a ts Rep78 or Rep68 protein. Also preferred are heat sensitive AAV Rep proteins. The amino terminus of the large Rep proteins are associated with DNA binding and other activities. Accordingly, illustrative AAV Rep proteins of the invention comprise a mutation in the amino-terminal half thereof, wherein the mutation confers a temperature sensitive phenotype to the AAV Rep protein. In a further exemplary embodiment, the AAV Rep protein comprises a mutation selected from the group consisting of: (a) a mutation at amino acid position 40, (b) a mutation at amino acid position 42, (c) a mutation at amino acid position 44, and (d) combinations of (a)-(c), wherein the mutation confers a temperature sensitive phenotype to the AAV Rep protein.
The invention further provides destabilizing mutations in the AAV Rep sequences that increase the turnover rate of the protein. In illustrative embodiments, the destabilizing mutation is selected from the group consisting of: (a) a missense mutation at the p19 start site (i.e., the translation start site for the Rep52 and Rep40 proteins), (b) a missense mutation at the 5xe2x80x2 splice donor site; and (c) a missense mutation at the p19 start site and 5xe2x80x2 splice donor site. These mutations may be further advantageously combined with the inventive ts mutations to provide another level of control over Rep activity.
As a further aspect, the present invention provides a nucleotide sequence encoding the inventive ts sensitive AAV Rep protein(s). In particular preferred embodiments, the sequence encodes both a ts AAV Rep78 protein and a temperature-sensitive AAV Rep68 protein. The nucleotide sequence may further encode the AAV Rep52 protein and/or an AAV Rep40 protein, which may also have a ts phenotype. In one particular embodiment, the invention provides a nucleotide encoding a ts Rep protein, wherein the nucleotide sequence comprises: (a) rep coding sequences encoding a temperature-sensitive AAV Rep78 protein and a temperature sensitive Rep68 protein, and (b) a rAAV template comprising a heterologous nucleotide sequence flanked by 5xe2x80x2 and 3xe2x80x2 AAV inverted terminal repeats, wherein the rep coding sequence is not flanked by the AAV inverted terminal repeats.
As another aspect, the present invention provides an AAV Rep protein comprising a mutation at amino acid position 412, wherein the AAV Rep protein is an AAV Rep78 or AAV Rep68 protein, and wherein the mutation results in a reduced (e.g., diminished or decreased) affinity of the AAV Rep protein for magnesium (e.g., at least about a 33%, 50%, 75%, 90%, 95% reduction or more). This mutant Rep protein may be regulated by modulating magnesium concentrations. This Rep protein also finds use for investigating the structure of the Rep protein magnesium pocket and the effects of magnesium concentration on Rep activity.
As a further aspect, the present invention provides a hybrid adenovirus or herpesvirus vector stably expressing a ts AAV Rep78 or ts Rep68 protein. Rep protein interference with the helper virus may be avoided at non-permissive temperatures. In other preferred embodiments, the hybrid helper virus expresses the AAV cap and rep genes, wherein the rep genes encode a ts AAV Rep78 and/or AAV Rep68 protein. In still other preferred embodiments, the adenovirus vector further comprises a rAAV template comprising a heterologous nucleotide sequence and an AAV inverted terminal repeat. This vector may be used to provide all of the functions necessary to package rAAV particles on a single construct.
As another preferred embodiment, the invention provides a hybrid adenovirus vector, comprising, in an adenovirus backbone: (a) the adenovirus 5xe2x80x2 and 3xe2x80x2 sequences sufficient for adenovirus replication and packaging; (b) AAV Rep coding sequences encoding a temperature-sensitive AAV Rep protein, wherein the AAV Rep protein is a Rep78 or Rep68 protein, and wherein the AAV Rep coding sequences are flanked by the adenoviral sequences of (a).
These hybrid helper vectors may be introduced into packaging cells along with a rAAV vector template (e.g., carried by a plasmid, viral vector, or embedded in the chromosomal DNA) to produce rAAV vectors. The hybrid vector may be amplified to produce a large-scale stock, and the stock used to infect a packaging cell in the presence of a rAAV template at the permissive temperature to induce AAV vector replication and packaging. In another preferred embodiment, the AAV vector is provided as a rAAV virus. Propagation of the hybrid helper virus and rAAV vector template as two viruses within a permissive cell provides rapid amplification of both the rAAV vector and the packaging machinery within the cell.
Accordingly, in one particular embodiment, the invention provides a method of producing a rAAV particle, comprising providing to a cell permissive for AAV replication: (a) a rAAV template comprising a (i) heterologous nucleotide sequence, and (ii) AAV packaging signal sequences sufficient for the encapsidation of the AAV template into infectious rAAV particles; and (b) AAV sequences sufficient for replication and packaging of the AAV template into infectious viral particles, wherein the AAV sequences encode a temperature sensitive AAV Rep protein, wherein the AAV Rep protein is a Rep78 or Rep68 protein; under conditions permissive for the temperature-sensitive AAV Rep protein and sufficient for the replication and packaging of the rAAV template, whereby infectious rAAV particles comprising the rAAV template are produced in the cell. The method may further comprise the step of collecting the infectious rAAV particles, e.g., from the medium and/or by lysing the cells. In particular embodiments, the method further comprises providing helper virus sequences which provide the helper virus functions essential for a productive AAV infection, wherein the helper virus sequences cannot be packaged into infectious rAAV viral particles. It is further preferred that the AAV replication and packaging sequences cannot be packaged into infectious rAAV viral particles.
As still a further aspect, the present invention provides improved methods of ex vivo gene delivery using AAV vectors. AAV is the only viral vector known to integrate at a specific target locus within the chromosome. Targeted integration is mediated by the Rep78 and/or Rep68 proteins. This unique attribute of AAV vectors has not been extensively utilized because the Rep proteins are absent from essentially all current ex vivo and in vivo gene therapy protocols. According to the present invention, the ts AAV Rep78 and/or Rep68 proteins are used to mediate targeted integration of AAV vectors in cells ex vivo at permissive temperatures. The cells may then be shifted to non-permissive temperatures to inactivate the ts Rep protein(s), thereby mitigating concerns regarding the presence of functional Rep protein in target cells as well as potential toxicity to target cells. The cells may then be administered to a subject in vivo, e.g., to produce a therapeutic or immunogenic response in the subject.
In one embodiment, the invention provides a method of integrating a nucleotide sequence into a chromosome of a cell, comprising: providing a nucleotide sequence to a cell, wherein the nucleotide sequence comprises a recombinant AAV template comprising (a) AAV sequences sufficient for integration of the AAV template into the cell, and (b) a heterologous nucleotide sequence, and providing a temperature-sensitive AAV Rep protein(s) to the cell, wherein the nucleotide sequence and the temperature-sensitive AAV Rep protein are provided under conditions permissive for the temperature-sensitive AAV Rep protein, whereby the recombinant AAV template is integrated into a chromosome of the cell.
In a further aspect of the invention, the modified cell is administered to a subject. In one particular embodiment, the invention provides a method of administering a nucleotide sequence to a subject, comprising: (a) providing a nucleotide sequence to a cell, wherein the nucleotide sequence comprises a recombinant AAV template comprising (a) AAV sequences sufficient for integration of the AAV template into the cell, and (b) a heterologous nucleotide sequence; and (b) providing to the cell a temperature-sensitive AAV Rep protein; wherein the nucleotide sequence and the temperature-sensitive Rep protein are provided under conditions permissive for the temperature-sensitive AAV Rep protein, whereby the recombinant AAV template is integrated into a chromosome of the cell; and (c) administering the cell to a subject. The method may further comprise the step of shifting the cell to a non-permissive temperature prior to administering the cell to the subject.
In particularly preferred embodiments of the present invention, the ts AAV Rep78 and/or Rep68 protein is expressed from a vector construct that also comprises a rAAV template to be delivered to the target cell. The Rep protein(s) mediates the targeted integration of the rAAV vector into the chromosome at the permissive temperature, but the Rep protein(s) are not integrated. This embodiment also advantageously permits the delivery of relatively large heterologous nucleotide sequences, which would exceed the limited capacity of the AAV capsid (approximately 4.5 kb), between the AAV ITRs. Preferably, the rep coding sequences are not flanked by the AAV integration sequences (e.g., ITRs).
These and other aspects of the present invention are described in more detail in the description of the invention set forth below.